Biomedical subjects

M Q Xu

Publications and source records attributed to M Q Xu.

At least 37 records · Page 2Linked to original sources

PubMed1997

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Affinity Labels↗

PubMed1997

Protein splicing and autoproteolysis mechanisms.

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

Animals↗

PubMed1996

The mechanism of protein splicing and its modulation by mutation.

Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.

Bacterial Proteins↗

PubMed1996

Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.

Amino Acid Sequence↗

PubMed1996

Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process.

Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein. A possible mechanism for branched intermediate formation is an N-O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate. This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp. GB-D. Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified. In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products. Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction. These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing.

Amino Acid Sequence↗

PubMed1995

Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence.

Protein splicing is a self-catalyzed, posttranslational process which converts a precursor polypeptide into two new proteins by the excision of an internal polypeptide segment and the ligation of the flanking polypeptides. Evidence has been presented that protein splicing involves a branched intermediate, which is resolved into the two protein products by the cyclization of an asparagine residue to aminosuccinimide [Xu, M. Q., Comb, D. G., Paulus, H., Noren, C. J., Shao, Y., & Perler, F. (1994) EMBO J. 13, 5517-5522]. This report describes the chemical synthesis of a peptide with a C-terminal aminosuccinimide residue, corresponding to the putative C-terminus of the excised intervening sequence (intein) derived from the thermostable DNA polymerase of Pyrococcus species GB-D. The synthetic aminosuccinimide peptide was compared with the C-terminal cyanogen bromide peptide of the excised intein and found to be indistinguishable in terms of its chromatographic properties, high-resolution mass spectrum, and colorimetric assay involving reaction with hydroxylamine. This establishes definitively that protein splicing is accompanied by the cyclization of asparagine to yield an aminosuccinimide residue at the C-terminus of the excised intein and that this unusual residue is therefore a natural constituent of spliced proteins. The effects of pH and temperature on the stability of the synthetic aminosuccinimide peptide are described.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

IPEB transcription factor regulating the intracisternal A particle gene during F9 cell differentiation is expressed at sites of lymphoid development.

The murine intracisternal A particle (IAP) proviral elements are expressed at low levels in undifferentiated F9 embryonal carcinoma cells but are highly expressed when F9 cells are induced to differentiate into parietal endoderm-like cells. IAP elements are also expressed in parietal endoderm-like PYS-2 cells. We previously identified an IAP proximal enhancer (IPE) element that mediates a F9 differentiation-specific enhancer activity. We also identified a 60 kDa IPE binding (IPEB) protein whose activity is high in PYS-2 cells, where IAP is expressed, but very low in F9 cells. Transcription of IAP elements has also been shown in the adult mouse thymus and in activated splenic B cells. We have now shown by DNA affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and band-shift analysis that the 60 kDa IPEB is expressed in adult T lymphocytes and in resting as well as lipopolysaccharide activated splenic B cells but not in adult liver cells, suggesting an important role for IPEB in IAP transcription in vivo. In addition, we find IPEB expressed in the fetal mouse at sites of lymphoid development, such as the liver, spleen, and thymus, suggesting it may play an important role in gene expression during lymphoid development. In support of this, we find IPEB in the human T cell tumor lines, Jurkat and Molt 13, as well as the Daudi B cell line and in the normal calf thymus and in the thymus and spleen of the chicken and rat.

Animals↗

PubMed1994

Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.

Alkalies↗

PubMed1994

Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase.

The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron. Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase. With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.

Amino Acid Sequence↗

PubMed1993

In vitro protein splicing of purified precursor and the identification of a branched intermediate.

Protein splicing is a posttranslational processing event in which an internal polypeptide is excised from a protein precursor and the terminal polypeptides are then ligated together, resulting in the production of two proteins. This report presents direct evidence for protein splicing by demonstrating in vitro splicing of purified precursor that accumulated when the protein splicing element from Pyrococcus DNA polymerase was cloned into a foreign gene. In vitro splicing was temperature and pH dependent. A slowly migrating species exhibited kinetic properties of a splicing intermediate and was shown to be a branched molecule by N-terminal sequencing. The precursor and slowly migrating species were interconvertible in response to pH shifts.

Amino Acid Sequence↗

PubMed1993

[Clinical analysis of 1314 cases with hepatic hydatidosis].

One thousand three hundred and fourteen cases of hepatic hydatidosis confirmed by surgical operation were analysed. Most of them contracted this disease in childhood, and hydatid cysts developed slowly without eliciting distinct symptoms until the occurrence of complications. The chief complaint was abdominal mass with pain in the right hypochondrium. The main complications in 1,314 patients were shown to be secondary bacterial infection of hydatid cysts (20.2%), cyst rupture (9.9%), portal hypertension (2.4%) and anaphylactic reaction (1.9%). By employing the advanced diagnostic technique especially the combined use of ultrasonography, roentgenography, CT scanning and seroimmunological diagnosis, the percentage of preoperative diagnosis might be raised to as high as 100%.

Adolescent↗

PubMed1992

Activation of the catalytic core of a group I intron by a remote 3' splice junction.

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.

Base Sequence↗

PubMed1992

A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.

Adenovirus E1A Proteins↗

PubMed1992

Diagnosis and management of hepatic hydatidosis complicated with biliary fistula.

A total of 1,204 patients with liver hydatidosis were treated by operations in our hospital from 1953 to 1990. Of these 74 had biliary fistulae. Growth of echinococcus cyst causes displacement, distortion and stenosis of the hepatic ductules with impaired bile drainage. Biliary effusion may occur between the endo- and ecto-cyst walls. Long term compression renders the hepatic ductule atrophic, and liable to rupture, forming a hydatid cyst-biliary fistula. The hydatid cyst can rupture into the biliary tract, and cyst fluid escapes into the biliary tract with daughter cysts discharged into the common bile duct, causing biliary colic, obstructive jaundice and possibly liver abscess. For acute obstructive and suppurative cholangitis, drainage of purulent bile and daughter cysts and management of the infected hydatid cyst are indicated. After removal of the echinococcus cyst, the fistulous opening on the hepatic duct must be sutured, but a small biliary fistula may be left alone. According to the thickness of the ectocyst wall, size of the cavity, severity of the infection, and degree of bile leakage, one of the following operative procedures for obliteration of the residual cavity can be selected: (1) closure by inversion suture of ectocyst; (2) omental or muscle flap obliteration; (3) closed catheter drainage.

Biliary Fistula↗

PubMed1990

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria.

A self-splicing group I intron has been found in the gene for a leucine transfer RNA in two species of Anabaena, a filamentous nitrogen-fixing cyanobacterium. The intron is similar to one that is found at the identical position in the same transfer RNA gene of chloroplasts of land plants. Because cyanobacteria were the progenitors of chloroplasts, it is likely that group I introns predated the endosymbiotic association of these eubacteria with eukaryotic cells.

Animals↗

PubMed1990

A self-splicing group I intron in the DNA polymerase gene of Bacillus subtilis bacteriophage SPO1.

We report a self-splicing intron in bacteriophage SPO1, whose host is the gram-positive Bacillus subtilis. The intron contains all the conserved features of primary sequence and secondary structure previously described for the group IA introns of eukaryotic organelles and the gram-negative bacteriophage T4. The SPO1 intron contains an open reading frame of 522 nucleotides. As in the T4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in a highly conserved region of the intron core. The exons encode SPO1 DNA polymerase, which is highly similar to E. coli DNA polymerase I. The demonstration of self-splicing introns in viruses of both gram-positive and gram-negative eubacteria lends further evidence for their early origin in evolution.

Amino Acid Sequence↗

PubMed1990

Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns.

The catalytic core of the sunY intron of bacteriophage T4 is separated from its 3' exon by 837 nucleotides, most of which are part of an open reading frame (ORF). Here, we report that transcripts truncated within the sunY ORF self-splice in vitro to a variety of sites in the segment immediately 3' of the core. Recognition of these proximal splice sites is shown to depend on (1) the presence on the intron side of a terminal G, which must not be part of a secondary structure; and (2) the ability of the penultimate intron nucleotide to base-pair with a 3' splice site-binding sequence (3'SSBS) located within the core. The counterpart of the 3'SSBS can be identified in most group I introns. The possible significance of such alternative splicing events for in vivo expression of intron-encoded proteins is discussed.

Base Composition↗

PubMed1989

The catalytic core of the sunY intron of bacteriophage T4.

The self-splicing sunY intron of bacteriophage T4 shares a common secondary structure with other group I introns. A long open reading frame within the intron is entirely 3' of the structural elements conserved in all group I introns. This catalytic core is the smallest yet described for a self-splicing intron. An internal deletion of 728 nucleotides (nt), leaving 196 nt at the 5' end and 109 nt at the 3' end, allows normal self-splicing. Transcripts terminating 196 nt 3' of the 5' splice site retain catalytic activity.

Catalysis↗